PROJECT SUMMARY Oligo- and polysaccharides participate in fundamental molecular interactions, and their location on cell surfaces makes them ideal candidates as biomarkers for various disease states. Heparan sulfate (HS) is a highly sulfated linear polysaccharide that participates in an assortment of cellular signaling events that are either advantageous or pathogenic, depending upon the sulfation pattern along the polysaccharide chain. High-affinity binding probes, such as antibodies, are an effective means for discerning molecular interactions within cells; however, few reagents are sufficiently specific for monitoring the sulfation patterns of HS in situ. This project will develop a new class of reagents that is capable of recognizing specific sulfation patterns within the HS chain by exploiting the inherent specificity of enzymes (sulfotransferases) that are responsible for transferring the sulfate groups to the polysaccharide during HS biosynthesis. Protein scaffolds will be created by catalytically inactivating the sulfotransferases, and the affinity of the scaffolds will be enhanced via computationally-guided single point mutations, as well as by directed evolution. By selectively targeting either the substrate or product of the enzymatic reaction, each inactive sulfotransferase has the potential to bind to two specific sequences of HS. This project will focus on sulfotransferases HS 2-O-Sulfotransferase (HS2ST) and 3-O-Sulfotransferase Isoform 1 (HS3ST-1); however, there are numerous isoforms of various enzymes from the biosynthetic pathway of HS that could be targeted in future studies. Both enzymes interact with biologically significant sulfation patterns. For example, HS2ST has been implicated in a variety of functions including angiogenesis, axon guidance, and prostate cancer. The HS3ST-1 enzyme is directly responsible for the anticoagulant properties of heparin, by increasing the affinity of HS for antithrombin. These reagents would be immensely useful for tracking changes in sulfation patterns via typical antibody-mediated assays, including affinity purification, western blotting, in situ histological staining, and in vivo imaging.